WO2001007452A1 - Extraction of nucleic acids - Google Patents

Extraction of nucleic acids Download PDF

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Publication number
WO2001007452A1
WO2001007452A1 PCT/GB2000/002842 GB0002842W WO0107452A1 WO 2001007452 A1 WO2001007452 A1 WO 2001007452A1 GB 0002842 W GB0002842 W GB 0002842W WO 0107452 A1 WO0107452 A1 WO 0107452A1
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WO
WIPO (PCT)
Prior art keywords
nucleic acids
liquid
positively charged
tube
electric field
Prior art date
Application number
PCT/GB2000/002842
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French (fr)
Other versions
WO2001007452A8 (en
Inventor
Matthew John Baker
Original Assignee
Dna Research Innovations Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Dna Research Innovations Limited filed Critical Dna Research Innovations Limited
Priority to AU62978/00A priority Critical patent/AU6297800A/en
Publication of WO2001007452A1 publication Critical patent/WO2001007452A1/en
Publication of WO2001007452A8 publication Critical patent/WO2001007452A8/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis

Definitions

  • the present invention relates to a method of isolating nucleic acids, particularly DNA, from biological mixtures.
  • nucleic acids The isolation and amplification of nucleic acids is used in many applications, for example in the detection and diagnosis of genetic and infectious disease, in forensic medicine, in the identification of new genes or allelic variations or mutations and in aiding routine genetic manipulation e.g. sequencing.
  • Amplification techniques such as polymerase chain reaction (PCR) are widely used and have proved very useful in a range of applications, however PCR cannot be used directly on clinical samples as the samples contain substances which inhibit the amplification enzyme and the presence of red blood cells presents a particular problem.
  • PCR polymerase chain reaction
  • nucleic acids are isolated from the samples before amplification etc.
  • a known method in which nucleic acids may be separated by charge differences uses gel electrophoresis. This is performed by adding the sample to a well in the gel and applying a voltage e.g. 100 volts, 60amps, through the gel, the nucleic acids gradually migrate through the gel depending on their molecular size. This method of separation is intended for analytical techniques relating to molecular size rather than extraction, since the nucleic acids are retained in the gel and would require further purification.
  • the present invention provides an improved method for separating of nucleic acids by electrical charge without using a gel.
  • a method for separating nucleic acids from liquid mixtures containing them which method comprises forming a positively charged surface in the liquid and forming an electric field through the liquid to cause nucleic acids to migrate towards the positive charged surface and binding the nucleic acids to the positively charged surface.
  • the invention also provides a method for separating nucleic acids from liquid mixtures containing them which method comprises passing an electric current through the liquid to form a positively charged surface in contact with the liquid to cause nucleic acids to migrate towards the positive charged surface and binding the nucleic acids to the positively charged surface.
  • the positively charged surface is formed, to which the nucleic acids migrate and become bound so that, after period of time, the surface can be removed and the nucleic acids separated.
  • the nucleic acids having a negative charge at physiological pH e.g. above pH 5 for example pH 5 to 9, will be attracted to a positively charged surface, and preferably the liquid is preferably buffered to maintain it at physiological pH. After washing away contaminants, the nucleic acids can be removed from this surface for analysis using PCR or other techniques. The nucleic acids can be released by turning off the current or reversing the polarity or by washing.
  • the nucleic acids bind to the positively charged surface by virtue of their negative charge, and the force binding them to the surface can be the electrical attraction between the negatively charged nucleic acids and the positively charged surface.
  • a sample containing cells is diluted in a lysing reagent that releases the nucleic acids into a liquid and a current or potential difference (i.e. an electric field) is then applied through the liquid using a technique in which a surface with a positive charge is generated.
  • a current or potential difference i.e. an electric field
  • the nucleic acids When an electric field is generated across the tube or vessel containing the liquids in which there are the nucleic acids, the nucleic acids will then move to the sides of the tube or vessel according to the local flux.
  • the electric field can be applied from outside the tube or vessel.
  • An electric current can be generated by having electrodes in contact with the liquid and the voltage can be in the order of 20 volts e.g. 5 to 50 volts so that an electric field is set up between the electrodes.
  • the electrodes can be in any convenient form, for example they can be made from an inert metal or conductive plastics material such as a plastic containing conductive particles such as graphite. This electrode can be made from an inert plastics material such as a polypropylene.
  • the conductive material can be inco ⁇ orated in a filter, membrane or pipette tip etc. through or across which the liquid containing the nucleic acids is passed so that the nucleic acids become bound to the filter, membrane or pipette tip etc.
  • An electrode e.g. metal electrode can be covered with a porous plastic material such as dialysis tubing so that the liquid forms a conductive path through the tubing, the nucleic acids then migrate to and become bound or attached to the tubing whilst the electric current is flowing and the bound nucleic acids can subsequently be removed.
  • a porous plastic material such as dialysis tubing
  • a pipette incorporates a conducting tip through which an electric current can be conducted, in this case the conducting pipette tip can be used to bind DNA by either having a two pipettes in the same sample vessel or tube and passing a current between them, so the nucleic acids will become bound to the positively charged pipette tip.
  • the sample tube can be modified e.g. it is modified with metal electrodes or the sample tube has conducting material inco ⁇ orated in the tube itself so that current is passed through liquid with the pipette tip as the positive electrode.
  • a conducting material may be included in a PCR tube or plate such as the wells in a 96 hole plate. If the PCR tube or plate is made positive relative to a negative electrode or tip dipping in the solution containing the nucleic acids, the nucleic acids will bind to the tube walls or plate, allowing the contaminants to be washed away. The bound nucleic acids may then be released by switching the charge off and the DNA is ready for PCR in the same tube.
  • the invention can be performed in a variety of formats for large or small samples and in an automated system.
  • the invention is particularly useful for separating nucleic acids from liquids which contain other material such as cell debris obtained from lysing cells, etc. which is not negatively charged.
  • One embodiment of the invention is the isolation of DNA from whole blood as described in the example.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • Analytical Chemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
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  • Biophysics (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
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Abstract

A method of isolating nucleic acids from liquid mixtures comprises passing an electric current through the liquid so as to form a positively charged surface to which the nucleic acids become bound and from which they can subsequently be removed.

Description

Extraction of Nucleic Acids
The present invention relates to a method of isolating nucleic acids, particularly DNA, from biological mixtures.
The isolation and amplification of nucleic acids is used in many applications, for example in the detection and diagnosis of genetic and infectious disease, in forensic medicine, in the identification of new genes or allelic variations or mutations and in aiding routine genetic manipulation e.g. sequencing.
Amplification techniques such as polymerase chain reaction (PCR) are widely used and have proved very useful in a range of applications, however PCR cannot be used directly on clinical samples as the samples contain substances which inhibit the amplification enzyme and the presence of red blood cells presents a particular problem.
In order to overcome this problem the nucleic acids are isolated from the samples before amplification etc.
A known method in which nucleic acids may be separated by charge differences uses gel electrophoresis. This is performed by adding the sample to a well in the gel and applying a voltage e.g. 100 volts, 60amps, through the gel, the nucleic acids gradually migrate through the gel depending on their molecular size. This method of separation is intended for analytical techniques relating to molecular size rather than extraction, since the nucleic acids are retained in the gel and would require further purification.
The present invention provides an improved method for separating of nucleic acids by electrical charge without using a gel. According to the invention there is provided a method for separating nucleic acids from liquid mixtures containing them which method comprises forming a positively charged surface in the liquid and forming an electric field through the liquid to cause nucleic acids to migrate towards the positive charged surface and binding the nucleic acids to the positively charged surface.
The invention also provides a method for separating nucleic acids from liquid mixtures containing them which method comprises passing an electric current through the liquid to form a positively charged surface in contact with the liquid to cause nucleic acids to migrate towards the positive charged surface and binding the nucleic acids to the positively charged surface.
The positively charged surface is formed, to which the nucleic acids migrate and become bound so that, after period of time, the surface can be removed and the nucleic acids separated.
The nucleic acids, having a negative charge at physiological pH e.g. above pH 5 for example pH 5 to 9, will be attracted to a positively charged surface, and preferably the liquid is preferably buffered to maintain it at physiological pH. After washing away contaminants, the nucleic acids can be removed from this surface for analysis using PCR or other techniques. The nucleic acids can be released by turning off the current or reversing the polarity or by washing.
The nucleic acids bind to the positively charged surface by virtue of their negative charge, and the force binding them to the surface can be the electrical attraction between the negatively charged nucleic acids and the positively charged surface.
In one embodiment of the method of the invention a sample containing cells is diluted in a lysing reagent that releases the nucleic acids into a liquid and a current or potential difference (i.e. an electric field) is then applied through the liquid using a technique in which a surface with a positive charge is generated.
When an electric field is generated across the tube or vessel containing the liquids in which there are the nucleic acids, the nucleic acids will then move to the sides of the tube or vessel according to the local flux. The electric field can be applied from outside the tube or vessel.
An electric current can be generated by having electrodes in contact with the liquid and the voltage can be in the order of 20 volts e.g. 5 to 50 volts so that an electric field is set up between the electrodes.
The electrodes can be in any convenient form, for example they can be made from an inert metal or conductive plastics material such as a plastic containing conductive particles such as graphite. This electrode can be made from an inert plastics material such as a polypropylene. The conductive material can be incoφorated in a filter, membrane or pipette tip etc. through or across which the liquid containing the nucleic acids is passed so that the nucleic acids become bound to the filter, membrane or pipette tip etc.
An electrode e.g. metal electrode can be covered with a porous plastic material such as dialysis tubing so that the liquid forms a conductive path through the tubing, the nucleic acids then migrate to and become bound or attached to the tubing whilst the electric current is flowing and the bound nucleic acids can subsequently be removed.
In an embodiment of the invention a pipette incorporates a conducting tip through which an electric current can be conducted, in this case the conducting pipette tip can be used to bind DNA by either having a two pipettes in the same sample vessel or tube and passing a current between them, so the nucleic acids will become bound to the positively charged pipette tip. Alternatively the sample tube can be modified e.g. it is modified with metal electrodes or the sample tube has conducting material incoφorated in the tube itself so that current is passed through liquid with the pipette tip as the positive electrode.
For very small samples, where a small quantity of nucleic acids is present, e.g. viral RNA from serum, a conducting material may be included in a PCR tube or plate such as the wells in a 96 hole plate. If the PCR tube or plate is made positive relative to a negative electrode or tip dipping in the solution containing the nucleic acids, the nucleic acids will bind to the tube walls or plate, allowing the contaminants to be washed away. The bound nucleic acids may then be released by switching the charge off and the DNA is ready for PCR in the same tube.
Thus the invention can be performed in a variety of formats for large or small samples and in an automated system.
The invention is particularly useful for separating nucleic acids from liquids which contain other material such as cell debris obtained from lysing cells, etc. which is not negatively charged.
One embodiment of the invention is the isolation of DNA from whole blood as described in the example.
Example
3 ml of whole blood was mixed with 30ml of lOmM Ammonium bicarbonate and 5% Tween 20 with lOmM Tris HCl pH 9, proteinase K 200 μg/ml. To form a buffered solution containing nucleic acids lysed from the blood cells. Conductive electrodes made of copper were covered with dialysis tubing containing the same buffer without the protease and dipped into the diluted sample. A power unit was used to apply a voltage of 20V between the electrodes and then left to run for 1 hour. The positive electrode was then washed in the same buffer and the power switched off. It was found that nucleic acids from the blood had been separated from the liquid and bound to the positively charged electrode and the DNA was then released into a working buffer ready for PCR.

Claims

Claims
1. A method for separating nucleic acids from liquid mixtures containing them which method comprises forming a positively charged surface in the liquid and forming an electric field through the liquid to cause nucleic acids to migrate towards the positive charged surface and binding the nucleic acids to the positively charged surface.
2. A method as claimed in claim 1 in which an electric field is generated through the liquid containing the nucleic acids from outside the vessel containing the liquid.
3. A method as claimed in claim 1 which method comprises passing an electric current through the liquid to form a positively charged surface in contact with the liquid and to form an electric field through the liquid.
4. A method as claimed in claim 3 in which the electric current is generated between electrodes in contact with the liquid containing the nucleic acids.
5. A method as claimed in claim 4 in which the positive electrode is made from a conductive plastics material
6. A method as claimed in claim 5 in which the conductive plastics materials comprises an inert plastics material containing conductive particles.
7. A method as claimed in any one of the preceding claims in which a conductive material is incoφorated in a component through or across which the liquid containing the nucleic acids is passed and the conductive material forms the positive surface.
8. A method as claimed in any one of the preceding claims in which a conductive material is incoφorated in a pipette tip which conductive material comprises the electrode.
9. A method as claimed in claim 8 in which two pipettes are placed in a liquid containing nucleic acids in a vessel or tube and an electric current is passed between the pipettes so the nucleic acids become bound to the positively charged pipette tip.
10. A method as claimed in claim 8 in which the pipette is placed in a liquid containing nucleic acids in a vessel or tube and an electric current is passed between the pipette as the cathode and there is an anode spaced apart from the pipette tip so the nucleic acids become bound to the positively charged pipette tip.
11. A method as claimed in any one of claims 1 to 6 in which a conducting material is included in a PCR tube or plate which is made positive relative to a negative electrode or tip dipping in the solution containing the nucleic acids and the nucleic acids bind to the tube walls or plate.
12. A method as claimed in any one ofthe preceding claims in which the nucleic acid is separated from cells and in which a sample containing the cells is diluted in a lysing reagent that releases the nucleic acids and the current or electric field is then applied through the liquid so formed.
PCT/GB2000/002842 1999-07-27 2000-07-27 Extraction of nucleic acids WO2001007452A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU62978/00A AU6297800A (en) 1999-07-27 2000-07-27 Extraction of nucleic acids

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB9917469.0 1999-07-27
GBGB9917469.0A GB9917469D0 (en) 1999-07-27 1999-07-27 Extraction of nucleic acids

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WO2001007452A1 true WO2001007452A1 (en) 2001-02-01
WO2001007452A8 WO2001007452A8 (en) 2001-05-03

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7014744B2 (en) 2001-08-24 2006-03-21 Applera Corporation Method of purification and concentration using AC fields with a transfer tip
WO2020079220A1 (en) * 2018-10-18 2020-04-23 Danmarks Tekniske Universitet Methods for extraction of molecules

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5422272A (en) * 1993-07-14 1995-06-06 Andrew A. Papp Improvements to apparatus and method for electroporation
WO1997008293A1 (en) * 1995-08-25 1997-03-06 Scientific Generics Limited Release of intracellular material
WO1997034908A1 (en) * 1996-03-15 1997-09-25 Innova Gesellschaft Zur Entwicklung Und Vermarktung Innovativer Produkte Mbh Process and device for isolating nucleic acids
WO1997041219A1 (en) * 1996-04-26 1997-11-06 Scientific Generics Limited Electrode capture of nucleic acid
US5733442A (en) * 1990-12-07 1998-03-31 Shukla; Ashok K. Microdialysis/Microelectrodialysis system

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5733442A (en) * 1990-12-07 1998-03-31 Shukla; Ashok K. Microdialysis/Microelectrodialysis system
US5422272A (en) * 1993-07-14 1995-06-06 Andrew A. Papp Improvements to apparatus and method for electroporation
WO1997008293A1 (en) * 1995-08-25 1997-03-06 Scientific Generics Limited Release of intracellular material
WO1997034908A1 (en) * 1996-03-15 1997-09-25 Innova Gesellschaft Zur Entwicklung Und Vermarktung Innovativer Produkte Mbh Process and device for isolating nucleic acids
WO1997041219A1 (en) * 1996-04-26 1997-11-06 Scientific Generics Limited Electrode capture of nucleic acid

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7014744B2 (en) 2001-08-24 2006-03-21 Applera Corporation Method of purification and concentration using AC fields with a transfer tip
US7611614B2 (en) 2001-08-24 2009-11-03 Applied Biosystems, Llc Method of cell capture
WO2020079220A1 (en) * 2018-10-18 2020-04-23 Danmarks Tekniske Universitet Methods for extraction of molecules

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Publication number Publication date
GB9917469D0 (en) 1999-09-29
AU6297800A (en) 2001-02-13
WO2001007452A8 (en) 2001-05-03

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